Advances in the molecular understanding of blood group antigens make it possible to address long-standing problems in Transfusion Medicine that cannot be addressed by the hemagglutination. In the current proposal we will use molecular biology procedures to improve clinical services for patients who are transfusion dependent. During the past four years we have made significant progress toward resolution of these problems in three areas: expression of blood group antigens in cell lines; production of several noel monoclonal blood group antibodies (mabs) with specificities to human polymorphic antigens; and development of genotyping approaches that are applicable in the clinical laboratory for identification of a fetus at risk for hemolytic disease of the newborn and to genotype recently transfused patients. We will now focus on: (1) optimizing expression of other clinically significant antigens with the long-term goal of developing a rapid objective, automated system to detect and identify antibodies that are relevant in transfusion and development of an assay to predict the potential of an antibody to cause in vivo RBC destruction; (2) engineering IgG mabs to direct agglutinins for rapid screening of RBCs we plan to expand our mab panel by using new immunogens in wild type, transgenic, and knockout mice. We will use a new fusion partner that does not require EBV transformation, to produce human mab anti-D for diagnosis and prophylaxis from the hyperimmunized human donors and engineer novel mabs using reshuffling of phage anti-D; (3) transferring the protocols we have established for genotyping to the clinical laboratory and to develop ISO-9000 style Standards. Since NYBC has a wide diversity of blood donors and our assays have recently revealed new alleles, we will test a larger sample pol to establish the incidence of common and variant alleles in different ethnicities and concurrently determine the relationship between genotype and phenotype. Collectively, these goals will lead to improving the practices used in transfusion medicine.